Novel a-lipoic acid derivative and use thereof

ABSTRACT

A novel a-lipoic acid derivative represented by the following formula (I). It has tyrosinase inhibitory activity, melanin production inhibitory activity, and elastase inhibitory activity. (I) (In the formula, M represents a metal and A represents an amino acid residue bonded through the nitrogen atom.).

TECHNICAL FIELD

[0001] The present invention relates to novel α-lipoic acid derivatives,pharmacologically acceptable salts thereof and applications thereof.

BACKGROUND ART

[0002] Alf-lipoic acid (also known as thioctic acid or6,8-dithiooctanoic acid), a coenzyme occurring in mitochondria, hasanti-oxidative activity and draws attention as a therapeutic agent for avariety of pathologic conditions induced by oxidative stress, such asarteriosclerosis and cataract. Its reduced state compound,dimercaptooctanoic acid, acts to restore reduced forms of glutathione orvitamin C back from their oxidized forms.

[0003] A class of α-lipoic acid derivatives, α-lipoylamino acids, inwhich α-lipoic acid is bound to glycine, methionine, glutamic acid,valine or the like, respectively are known (Japanese Patent PublicationNo. S42-1286, and its corresponding U.S. Pat. No. 3,238,224). JapanesePatent Application Publication No. 2000-169371 discloses a salt ofα-lipoylaminoethylsulfonic acid with imidazole and its use as anenhancer of glutathione reductase activity.

[0004] Upon the above background, the present inventors succeeded inefficient synthesis of metal chelate compounds and theirpharmacologically acceptable salts of reduced forms (dihydro-forms) ofnovel α-lipoylamino acids, found that these compounds possess atyrosinase inhibiting activity, a melanin production suppressingactivity and an elastase inhibiting activity, and completed the presentinvention through further studies.

DISCLOSURE OF INVENTION

[0005] Thus, the present invention relates to:

[0006] (1) an N-(6,8-dimercaptooctanoyl)amino acid metal chelatecompound represented by the following formula (I),

[0007] wherein M denotes a metal, and A denotes an amino acid which isbound via N, or a pharmacologically acceptable salt thereof (hereinafterreferred to as the present compound),

[0008] (2) the N-(6,8-dimercaptooctanoyl)amino acid metal chelatecompound of the above-identified formula (I) or a pharmacologicallyacceptable salt thereof, wherein the N-(6,8-dimercaptooctanoyl)aminoacid metal chelate compound is selected from the group consisting ofN-(6,8-dimercaptooctanoyl)-α-amino acid metal chelates,N-(6,8-dimercaptooctanoyl)-β-amino acid metal chelates,N-(6,8-dimercaptooctanoyl)-γ-amino acid metal chelates,N-(6,8-dimercaptooctanoyl)-δ-amino acid metal chelates, andN-(6,8-dimercaptooctanoyl)-ε-amino acid metal chelates,

[0009] (3) the N-(6,8-dimercaptooctanoyl)amino acid metal chelatecompound of the above-identified formula (I) or a pharmacologicallyacceptable salt thereof, wherein the N-(6,8-dimercaptooctanoyl)aminoacid metal chelate compound is selected from the group consisting ofN-(6,8-dimercaptooctanoyl)-aminoethanesulfonic acid metal chelates,N-(6,8-dimercaptooctanoyl)glycine metal chelates,N-(6,8-dimercaptooctanoyl)aspartic acid metal chelates,N-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid metal chelate,N-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid metal chelates,N-(6,8-dimercaptooctanoyl)-phenylalanine metal chelates,N-(6,8-dimercaptooctanoyl)anthranilic acid metal chelates,N-(6,8-dimercaptooctanoyl) methionine metal chelates, andN-(6,8-dimercaptooctanoyl)cysteine metal chelates,

[0010] (4) the N-(6,8-dimercaptooctanoyl)amino acid metal chelatecompound of the above-identified formula (I) wherein the metal is zinc,or a pharmacologically acceptable salt thereof,

[0011] (5) a medicament comprising the compound or a pharmacologicallyacceptable salts thereof defined in one of (1)-(4) above,

[0012] (6) a tyrosinase inhibiting agent comprising the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0013] (7) a melanin production suppressing agent comprising thecompound or a pharmacologically acceptable salt thereof defined in oneof (1)-(4) above,

[0014] (8) a prophylactic or therapeutic agent for blotches and frecklesor a suntan of the skin comprising the compound or a pharmacologicallyacceptable salt thereof defined in one of (1)-(4) above,

[0015] (9) a whitening agent comprising the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0016] (10) a skin-beautifying agent comprising the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0017] (11) an elastase inhibiting agent comprising the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0018] (12) an anti-wrinkle agent comprising the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0019] (13) a cosmetic preparation comprising the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0020] (14) a method for inhibition of tyrosinase comprisingadministering to a human an effective amount of the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0021] (15) a method for suppression of melanin production comprisingadministering to a human an effective amount of the compound or apharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0022] (16) a method for prophylaxis or therapeutic treatment ofblotches and freckles or a suntan of the skin comprising administeringto a human an effective amount of the compound or a pharmacologicallyacceptable salt thereof defined in one of (1)-(4) above,

[0023] (17) a method for whitening the skin comprising administering toa human an effective amount of the compound or a pharmacologicallyacceptable salt thereof defined in one of (1)-(4) above,

[0024] (18) a method for beautifying the skin comprising administeringto a human an effective amount of the compound or a pharmacologicallyacceptable salt thereof defined in one of (1)-(4) above,

[0025] (19) a method for inhibiting elastase comprising administering toa human an effective amount of the compound or a pharmacologicallyacceptable salt thereof defined in one of (1)-(4) above,

[0026] (20) a method for prevention or therapeutic treatment of wrinklescomprising administering to a human an effective amount of the compoundor a pharmacologically acceptable salt thereof defined in one of (1)-(4)above,

[0027] (21) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of amedicament,

[0028] (22) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of atyrosinase inhibiting agent,

[0029] (23) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of a melaninproduction suppressing agent,

[0030] (24) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of aprophylactic or therapeutic agent for blotches and freckles or a suntanof the skin,

[0031] (25) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of awhitening agent,

[0032] (26) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of askin-beautifying agent,

[0033] (27) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of aelastase inhibiting agent,

[0034] (28) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of ananti-wrinkle agent, and

[0035] (29) use of the compound or a pharmacologically acceptable saltthereof defined in one of (1)-(4) above for the manufacture of acosmetic preparation.

[0036] The present compound is of a structure consisting of α-lipoicacid that is amide-bonded with an amino acid and a chelated metaltherewith, and is a novel compound that has not been found in aliterature. In the present invention, amino acid means α-amino acid,β-amino acid, γ-amino acid, δ-amino acid and ε-amino acid, which have intheir molecule a carboxyl group together with an amino group, andaminomethylcyclohexanoic acid and anthranilic acid, as well asaminoethanesulfonic acid (taurine), which has in the molecule a sulfonicacid group together with an amino group. Examples of α-amino acidsinclude glycine, alanine, valine, leucine, isoleucine, serine,threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine,glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine,tryptophan, etc. Examples of β-amino acids include β-alanine. Examplesof γ-amino acids include γ-amino-n-butyric acid (GABA) and carnitine.Examples of δ-amino acids include 5-aminolevulinic acid, and5-aminovaleric acid. Examples of ε-amino acids include 6-aminohexanoicacid. Among those amino acids, anthranilic acid, aminoethanesulfonicacid, methionine, phenylalanine, γ-amino-n-butyric acid and6-aminohexanoic acid are preferred.

[0037] As a metal in the metal chelate compounds of the presentinvention, preferably employed are zinc and cobalt, which are divalentmetals, iron, which is a divalent or trivalent metal, and tetravalentgermanium. Among them, zinc, which is a divalent metal, is particularlypreferred. The present compound is an easily purified, stable compound.

[0038] Examples of pharmacologically acceptable salts of the presentcompound include alkali metal salts such as sodium salt and potassiumsalt and alkali earth metal salts such as calcium salt and magnesiumsalt. Any other salts may be employed as desired for the purpose of thepresent invention insofar as they are pharmacologically acceptable.

[0039] Further, monohydrates, dihydrates, 1/2 hydrates, 1/3 hydrates,1/4 hydrates, 2/3 hydrates, 3/2 hydrates and 6/5 hydrates are alsoincluded in the present invention.

[0040] In general, for the synthesis of α-lipoylamino acids,intermediate compounds, an amino acid is esterified on its carboxylgroup to get the group protected, then converted to an acid amide withα-lipoic acid using a dehydration condensation agent, and finallysubjected to saponification. However, synthesis is difficult by thismethod where aminoethanesulfonic acid is employed.

[0041] The present compound may be synthesized as desired by, oraccording to, e.g., the following scheme of synthesis.

[0042] As a result of a study for an efficient production ofα-lipoylamino acid, an intermediate for the present compound, it wasfound that the aimed α-lipoylamino acid is obtained in high yield whensynthesized by the mixed acid anhydride method (MA). Briefly, α-lipoicacid dissolved in an organic solvent (e.g., chloroform, tetrahydrofuran,acetonitrile, etc.) is reacted with a mixed acid anhydride reagent suchas a halogenated carbonic acid ester (such as ethyl carbonyl chloride,butyl carbonyl chloride), isobutyloxycarbonyl chloride, diethylacetylchloride, trimethylacetyl chloride and the like to form an mixed acidanhydride of α-lipoic acid at −15 to −5° C. in the presence of tertiaryamine (such as triethylamine, tributylamine, N-methylmorpholine (NMM) ).The length of time of the reaction is from 1-2 minutes to several tensof minutes. Then, an amino acid dissolved in alcohol, water or a mixturesolution thereof in the presence of a base (such as sodium hydroxide,potassium hydroxide, or a tertiary amine such as triethylamine ortributylamine) is added and reacted. Subsequent recrystallization from asuitable solvent, e.g., water or alcohol, gives α-lipoylamino acid inhigh yield.

[0043] Reduction of thus obtained α-lipoylamino acid with a metal and anacid gives, via a dihydro compound, a chelate compound of the presentinvention in high yield. Examples of acids employed in the reduction ofa stable α-lipoylamino acid includes inorganic acids such ashydrochloric acid and sulfuric acid and organic acids such as aceticacid and citric acid. In the case of a zinc chelate compound, the two—SH groups (mercapto group) in its molecule are thought to bind with onezinc atom.

[0044] Melanin in the skin is produced by melanocytes. Tyrosinase haslong been known as a sole rate-controlling enzyme which regulatesmelanin production. Tyrosinase is catalyzed by three enzymes, i.e.,tyrosine hydroxylase, 3,4-dihydroxyphenylalanine (DOPA) oxidase and5,6-dihydroxyindole (DHI) oxidase, which play important roles in earlyas well as late reactions in melanin production. As evident from thetest examples mentioned below, the present compound suppresses melaninproduction by inhibiting tyrosinase, the rate-controlling enzyme, viainhibition of tyrosine hydroxylase.

[0045] On the other hand, elastase is an enzyme which splits elastin, anelastic structure protein having a number of cross links among itspeptide chains and occurring in tissues with considerable extensibilitysuch as the skin. Therefore, inhibition of elastase in the skin would beuseful for prevention of wrinkles and maintenance of the beauty of theskin, for it would serve to maintain the extensibility and elasticity ofthe skin. As evident from the test examples described below, the presentcompound also has an elastase inhibiting activity.

[0046] Thus, the present compound has, as its features, melaninproduction suppressing activity and elastase inhibiting activity.

[0047] Therefore, the present compound is useful as a prophylactic ortherapeutic agent for blotches and freckles or suntan of the skin, awhitening agent, a skin-beautifying agent and an anti-wrinkle agent.

[0048] The present compound also exhibits anti-oxidation activity andradical-suppressing activity (eliminating a stable radical,1,1-diphenyl-2-picrylhydroradical (DPPH)). That the present compound hassuch activity is demonstrated by the fact that one mole of the presentcompound is required to reduce and decolorize one mole of iodine (I₂),which is consistent with what is theoretically calculated, and thepresent compound itself is thereby oxidized back to α-lipoylamino acid(see the following scheme).

[0049] The present compound is useful for prophylaxis and treatment of avariety of disorders caused by oxidative stress in mammals (e.g.,bovine, horse, rabbit, mouse, rat, human), e.g., ischemic cardiovasculardisease, cerebral ischemia, arteriosclerosis, diabetes mellitus andcataract.

[0050] When the present compound is used as a medical drug, one of thespecies of the present compound, or two or more of them in combination,may be employed as desired according to purpose and need.

[0051] The present compound is used as desired orally or parenterally asa drug for the treatment of the aforementioned disorders. As forpreparation forms, it may be provided in any form by a known method,e.g., solid preparations such as tablets, granules, powder, capsules, orliquid preparations such as injection or eye drops. These preparationsmay contain conventional additives as desired, such as diluents,binders, thickening agents, dispersing agents, reabsorption enhancers,buffers, surfactants, solubilizing agents, preservatives, emulsifiers,isotonizers, stabilizers, pH adjusting agents, etc.

[0052] The doses of the present compound, when used as a medical drug,will vary according to the employed species of the present compound, thebody weight and age of a given patient, the disorder to be treated andits condition and the way of administration. When used for ischemiccardiovascular diseases, cerebral ischemia, arteriosclerosis or diabetesmellitus, the doses for an adult are about 1 mg to about 30 mg per daygiven at a time in the case of an injection and about 1 mg to about 100mg at a time, administered several times a day, in the case of an oralpreparation. When used as an anti-cataract drug, eye drops containingabout 0.01 to 5 (w/v) % are preferably applied several times a day,several drops at each time.

[0053] A medical drug containing the present compound may also containother ingredients having the same or different pharmacological activityinsofar as they do not act counter to the purpose of the presentinvention.

[0054] The compound of the present invention may be added as desired tocreams, facial masking agents, powders, lotions, toilet waters forprophylaxis or treatment of blotches and freckles or suntan of the skin,or for whitening, skin beatifying or anti-wrinkle activity. When thepresent compound is added to cosmetic products, other additivesconventionally used in cosmetic products may also be employed, such asdiluents, stabilizers, pigments, fragrant materials, ultravioletabsorbents, antioxidants, preservatives, metal chelating agents andorganic acids.

[0055] When the present compound is used for cosmetic products, it isadded usually at about 0.001 to 5 (w/w) %, preferably at about 0.005 to2 (w/w) %, although this depends on the employed species of thecompound, the type of the cosmetic product and the purpose of theaddition.

EXAMPLES

[0056] The present invention will be described below in further detailwith reference to the reference examples, examples and test examples.However, the scope of the present invention is not limited by them.

Reference Example 1 N-α-lipoylaminoethanesulfonic Acid Sodium Salt

[0057] To 6.2 g of DL-α-lipoic acid, dissolved in 60 ml of chloroform,was added 3.2 g of triethylamine, and cooled at −5° C. To this, 3.3 g ofethyl carbonyl chloride was slowly added dropwise and, 15 minutes afterthe completion of the addition, 4.5 g of aminoethanesulfonic acid and1.5 g of sodium hydroxide dissolved in 60 ml of methanol were added atonce, and stirred for 15 minutes at the same temperature and further onehour at room temperature. To this was added a solution of 1.5 g ofsodium hydroxide dissolved in 50 ml of methanol, the solvent wascondensed under reduced pressure to ⅓ of the initial volume, 60 ml ofethanol was added, and precipitated crystals were collected byfiltration. Recrystallized of this from water-methanol gave 5.8 g ofwhite crystals of aimed sodium salt. Melting point: 235-237° C. TLC,Rf=0.53 (n-butanol:acetic acid:water=4:1:2)

[0058] Elemental Analysis: For C₁₀H₁₇NO₄S₃Na.H₂O. Calculated: C, 34.08H, 5.43 N, 3.97. Found : C, 34.23 H, 5.54 N, 3.80.

Reference Example 2 N-α-lipoylaminoethanesulfonic Acid Potassium Salt

[0059] The same procedure as Reference Example 1 was followed exceptthat sodium hydroxide in Reference Example 1 was replaced with 4.0 g ofpotassium hydroxide. Recrystallization from water/methanol gave 6.5 g ofwhite crystals of aimed potassium salt. Melting point: 240-242° C.

Reference Example 3 N-α-lipoylaminoethanesulfonic Acid Calcium andMagnesium Salts

[0060] The sodium salt obtained in Reference Example 1 was dissolved inwater, desalted with a sulfonic acid type resin into a free acid form,and neutralized with calcium carbonate or basic magnesium carbonate togive soluble calcium and magnesium salts, respectively. Melting point:over 300° C., respectively.

Reference Example 4 N-α-lipoyl-6-aminohexanoic Acid Sodium Salt

[0061] Using 4.2 g of DL-α-lipoic acid and 3.0 g of 6-aminohexanoicacid, the reaction was conducted in the same manner as ReferenceExample 1. Recrystallization from ethanol gave 3.0 g of yellowish whitecrystals of the aimed compound. Melting point: 200-202° C. (decomp.).TLC, Rf=0.84 (chloroform:methanol:water=5:4:1)

Reference Example 5 N-α-lipoylaspartic Acid Disodium Salt

[0062] Using 4.2 g of DL-α-lipoic acid and 2.9 g of L-aspartic acid, thereaction was conducted in the same manner as Reference Example 1.Recrystallization from water/methanol gave 4.5 g of white crystals ofthe aimed compound. Melting point: over 300° C. TLC, Rf=0.47(chloroform:methanol:water=4:1:2)

Reference Example 6 N-α-lipoyl-γ-amino-n-butyric Acid Sodium Salt

[0063] Using 4.2 g of DL-α-lipoic acid and 2.3 g of γ-amino-n-butyricacid, the reaction was conducted in the same manner as ReferenceExample 1. Recrystallization from ethanol gave 4.0 g of sodium salt, theaimed compound. Melting point: gradual decomposition starting at about235° C. TLC, Rf=0.76 (chloroform:methanol:water=4:1:2)

Reference Example 7 N-α-lipoylglycine Sodium Salt

[0064] Using 4.2 g of DL-α-lipoic acid and 1.9 g of glycine, thereaction was conducted in the same manner as Reference Example 1.Recrystallization from methanol/ethanol gave 4.5 g of pale yellowcrystals of the aimed compound. Melting point: 218-220° C. (decomp.)TLC, Rf=0.64 (chloroform:methanol:water=4:1:2)

Reference Example 8 N-α-lipoylphenylalanine

[0065] Using 4.2 g of DL-α-lipoic acid and 3.5 g of phenylalanine, thereaction was conducted in the same manner as Reference Example 1. Afterevaporation of the solvent and acidification with hydrochloric acid,extraction was carried out with ethyl acetate. Following washing withwater, ethyl acetate was evaporated. The crystalline residue wasrecrystallized from ethanol/isopropyl ether, giving 5.4 g of pale yellowcrystals. Melting point: 154-156° C. TLC, Rf=0.86 (nbutanol:acetic acid:water=4:1:2)

Reference Example 9 N-α-lipoylanthranilic Acid Sodium Salt

[0066] Using 4.2 g of DL-α-lipoic acid and 2.9 g of anthranilic acid,the same procedure followed as Reference Example 1 gave 3.6 g of whitecrystals. Melting point: over 300° C. TLC, Rf=0.89 (n-butanol:aceticacid:water=4: 1:2)

Reference Example 10 N-α-lipoylmethionine

[0067] Using 4.2 g of DL-α-lipoic acid and 3.5 g of L-methionine, thesame procedure followed as Reference Example 8 gave 4.0 g of pale yellowcrystals. Melting point: 108-109° C. TLC, Rf=0.81 (n-butanol:aceticacid:water=4:1:2)

Reference Example 11 N-α-lipoylcysteine Sodium Salt

[0068] 4.2 g of DL-α-lipoic acid and 2.2 g of triethylamine weredissolved in 60 ml of tetrahydrofuran and cooled at −5 ° C. To this, 2.3g of ethyl carbonyl chloride was slowly added dropwise and, 6 minutesafter the completion of the addition, a solution of 2.6 g of L-cysteineand 2.5 g of triethylamine dissolved in 20 ml of water was added andreacted as in Reference Example 1. After acidification with hydrochloricacid, extraction was carried out with ethyl acetate. Following washing,ethyl acetate was evaporated, and the residue was dissolved in ethanol.By gradual addition of sodium hydroxide in methanol to adjust the pH to7, 4.3 g of precipitated white crystals were obtained. Melting point:gradual decomposition starting at about 150° C. TLC, Rf=0.72(n-butanol:acetic acid:water=4:1:2). For a product whereN-ethylmaleimide was added, Rf=0.69 was found.

Reference Example 12 N-α-lipoyl-5-hydroxytryptophan

[0069] Using 4.2 g of DL-α-lipoic acid and 5.0 g ofL-5-hydroxytryptophan, the same procedure followed as Reference Example8 gave 6.4 g of white crystals. Melting point: 118-120° C. TLC, Rf=0.85(n-butanol:acetic acid:water=4:1:2)

Example 1 N-(6,8-dimercaptooctanoyl)aminoethanesulfonic Acid Sodium SaltZinc Chelate Compound

[0070] 5.0 g of the compound in the form of sodium salt obtained inReference Example 1 was dissolved in 100 ml of water, and to this wereadded 10 ml of acetic acid and 1.3 g of zinc powder. After one-hourstirring at 50° C., unreacted zinc was filtered off, the filtrateconcentrated under reduced pressure, and ethanol was added. Precipitatedwhite crystals were collected by filtration, dissolved in water,adjusted of the pH to about 8 with sodium bicarbonate, and concentrated.Methanol then was added and precipitated white crystals were collectedby filtration. Recrystallization from water/methanol gave 4.3 g of theaimed compound. Melting point: decomposition starting at about 293° C.TLC, Rf=0.51 (n-butanol:acetic acid:water=:4:1:2) Elemental Analysis:For C₁₀H₁₇NO₄S₃NaZn.H₂O Calculated: C, 28.75 H, 4.58 N, 3.35 Found: C,28.56 H, 4.69 N, 3.13

Example 2 N-(6,8-dimercaptooctanoyl)aminoethanesulfonic Acid PotassiumSalt Zinc Chelate Compound

[0071] The same procedure as in Example 1 was followed using 6.5 g ofthe compound obtained in Reference Example 2, giving 5.0 g of the aimedcompound. Elemental Analysis: For C₁₀H₁₇NO₄S₃KZn.1/2H₂O Calculated: C,28.27 H, 4.27 N, 3.30 Found: C, 28.38 H, 4.52 N, 3.10

Example 3 N-(6,8-dimercaptooctanoyl)glycine Sodium Salt Zinc ChelateCompound

[0072] The same procedure as in Example 1 was followed using 4.5 g ofthe compound obtained in Reference Example 7, giving 3.9 g of the aimedcompound. Melting point: decomposition starting at about 297° C. TLC,Rf=0.64 (chloroform:methanol:water=4:1:2) Elemental Analysis: ForC₁₀H₁₆NO₃S₂NaZn.H₂O Calculated: C, 32.57 H, 4.92 N, 3.80 Found: C, 32.43H, 4.83 N, 3.74

Example 4 N-(6,8-dimercaptooctanoyl)aspartic Acid Sodium Salt ZincChelate Compound

[0073] Reduction was performed as in Example 1 using 3.0 g of thecompound obtained in Reference Example 5. Precipitated white crystalswere collected by filtration and suspended in water, dissolved at pH 7-8with sodium hydroxide, insoluble matter was filtered off, and filtrateconcentrated. Methanol was added and precipitated white crystals werecollected by filtration, giving 2.3 g of the aimed compound. Meltingpoint: decomposition starting at about 295° C. TLC, Rf=0.53(chloroform:methanol:water=5:4:1) Elemental Analysis: ForC₁₂H₁₇NO₅S₂NaZn.H₂O Calculated: C, 32.11 H, 4.29 N, 3.12 Found: C, 32.09H, 4.44 N, 3.10

Example 5 N-(6,8-dimercaptooctanoyl)-6-aminohexanoic Acid Sodium SaltZinc Chelate Compound

[0074] 3.0 g of the compound obtained in Reference Example 4 wasdissolved in 70 ml of 50% tetrahydrofuran and reduced as in Example 1.The solvent was evaporated and precipitated white crystals werecollected by filtration. Melting point: 215-217° C. These were suspendedin water, and dissolved at pH 7-8 with sodium hydroxide. The solutionwas concentrated and methanol was added. Precipitated white crystalswere collected by filtration, giving 2.0 g of the aimed compound.Melting point: decomposition starting at about 295° C. TLC, Rf=0.84(chloroform:methanol:water=5:4:1) Elemental Analysis: ForC₁₄H₂₄NO₃S₂NaZn.H₂O Calculated: C, 39.58 H, 6.17 N, 3.30 Found: C, 39.38H, 6.02 N, 3.13

Example 6 N-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric Acid Sodium SaltZinc Chelate Compound

[0075] 4.0 g of the compound obtained in Reference Example 6 was reducedand worked out as in Example 1, giving 2.1 g of white crystals of theaimed compound. Melting point: decomposition starting at about 297° C.TLC, Rf=0.70 (chloroform:methanol:water=5:4:1) Elemental Analysis: ForC₁₂H₂₀NO₃S₂NaZn.H₂O Calculated: C, 36.32 H, 5.59 N, 3.53 Found: C, 36.08H, 5.81 N, 3.29

Example 7 N-(6,8-dimercaptooctanoyl)phenylalanine Sodium Salt ZincChelate Compound

[0076] To 5.4 g of the compound obtained in Reference Example 8 wereadded 80 ml of 30% methanol, 2.0 g of zinc powder, 10 ml of acetic acidand 20 ml of 2 N HCl. After 3-hour stirring at 50° C., unreacted zincwas filtered off, and the filtrate was concentrated. Following additionof water, precipitated oily matter was collected. For conversion to asodium salt, this was dissolved in methanol and pH adjusted to 7 withsodium hydroxide/methanol. Precipitated crystals were collected byfiltration, giving 3.9 g. Melting point: gradual decomposition startingat about 270° C. TLC, Rf=0.82 (n-butanol:acetic acid:water=4:1:2)Elemental Analysis: For C₁₇H₂₃NO₃S₂NaZn.1/2H₂O Calculated: C, 45.39 H,5.15 N, 3.11 Found: C, 45.55 H, 5.33 N, 3.23

Example 8 N-(6,8-dimercaptooctanoyl)anthranilic Acid Sodium Salt ZincChelate Compound

[0077] The same procedure as Example 7 was followed using 3.6 g of thecompound obtained in Reference Example 9, giving 2.1 g of whitecrystals. Melting point: decomposition starting at about 290° C. TLC,Rf=0.88 (nbutanol:acetic acid:water=4:1:2) Elemental Analysis: ForC₁₅H₁₈NO₃S₂NaZn.H₂O Calculated: C, 41.81 H, 4.68 N, 3.25 Found: C, 41.98H, 4.64 N, 3.26

Example 9 N-(6,8-dimercaptooctanoyl)methionine Zinc Chelate Compound

[0078] The same procedure was followed as Example 7 using 4.0 g of thecompound obtained in Reference Example 10, giving 2.8 g of the aimedcompound, free acid. Melting point: gradual decomposition starting atabout 260° C. TLC, Rf=0.82 (n-butanol:acetic acid:water=4:1:2) ElementalAnalysis: For C₁₃H₂₃NO₃S₃Zn.1.5H₂O Calculated: C, 36.32 H, 6.10 N, 3.26Found: C, 36.17 H, 5.78 N, 3.34

Example 10 N-(6,8-dimercaptooctanoyl)cysteine Zinc Chelate Compound

[0079] To 5.7 g of the compound obtained in Reference Example 11 wereadded 100 ml of 50% methanol, 3.5 g of zinc powder, 10 ml of acetic acidand 40 ml of 2 N hydrochloric acid. After 3-hour stirring at 50° C.,unreacted zinc was filtered off, the filtrate concentrated to about ½.The pH of this was adjusted to 3-4 with 2 N sodium hydroxide andprecipitated white crystals were collected by filtration and washed with3% acetic acid and water. They were dissolved in 1% sodium hydroxide,acidified with acetic acid, and precipitated crystals was collected byfiltration, washed with water and methanol, and dried. Melting point:decomposition starting at about 280° C. TLC (after dissolving byneutralization with ammonium water), Rf=0.71(chloroform:methanol:water=5:4: 1) Elemental Analysis: ForC₂₂H₃₆N₂O₆S₆Zn.3H₂O Calculated: C, 31.79 H, 4.61 N, 3.37 Found: C, 31.98H, 4.77 N, 3.14

Test Example 1 Tyrosine Hydroxylase Inhibiting Activity of The PresentCompound

[0080] The present compound was examined for tyrosine hydroxylaseinhibiting activity.

Test Method

[0081] Tyrosine hydroxylase from rat brain was used in the test.

[0082] A test compound added to 100 μM tyrosine containing L-[3,5-³H]tyrosine (0.5 μCi/assay), 1 mg of tyrosine hydroxylase, 0.2 mM of(6R)-5,6,7,8-tetrahydro-L-biopterin, 1.8 mg/ml catalase, 5 mMdithiothreitol (in MES buffer, pH 6.0) was incubated at 37° C. for 40minutes. The reaction was terminated by addition of 7.5% charcoal in 1 Mhydrochloric acid.

[0083]³H₂O derived from 3,4-dihydroxyphenylalanine (DOPA) was determinedby liquid scintillation.

Test Compound

[0084] The compounds of Reference Example 1, Example 1, and α-lipoicacid (final concentration: 0.1 mM, 1 mM).

Test Results

[0085] The results are shown in Table 1. TABLE 1 Tyrosine HydroxylaseInhibiting Activity of The Present Compound Concentration (%) 0.1 mM 1mM Compound of Reference Example 1 11 10 Compound of Example 1 15 53α-lipoic acid 2 1

[0086] As evident from Table 1, tyrosine hydroxylase inhibiting activitywas noted with the present compound. In contrast, α-lipoic acidexhibited no tyrosine hydroxylase inhibiting activity. Thus, the presentcompound was found to inhibit tyrosinase, an enzyme regulating melaninproduction.

Test Example 2 Melanin Production Suppressing Activity of The PresentCompound Test Method

[0087] B16-F0 melanoma cell line of mouse origin, purchased fromDainippon Pharmaceutical Co., Ltd. was used in the experiment. In 60-mmpetri dish, 200,000 cells were incubated for five days in a medium(D-MEM*+10% FBS*) supplemented with 0.1% D-glucosamine hydrochloride, aninhibitor of carbohydrate synthesis, to halt melanin synthesis and turnthe culture white. Following the 5-day culture, the cells were washedwith PBS(−)* to remove D-glucosamine hydrochloride, and 2 mMtheophylline (250-fold concentrated solution/distilled water), aphosphodiesterase inhibitor, was added to raise intracellular cAMP andthereby promote the recovery of tyrosinase synthesis. At the same time,a test compound (250-fold concentrated solution/PBS(−)) was added. Threedays after the addition of theophylline and the test compound, the cellswere harvested by trypsin treatment. The cell pellets were resuspendedin 1 ml of PBS(−), 0.1 ml of which was used for counting cells, and 0.9ml for melanin determination. Determination of melanin was carried outby washing the cell pellets once with 5% trichloroacetic acid,ethanol/diethyl ether (3:1), and then diethyl ether, adding 1 ml of 2 NNaOH to lyse the cells, measuring optical density at 420 nm, andcomparing the result with a calibration curve produced with referencestandard.

[0088] *(NB)

[0089] D-MEW: Dulbecco's Modified EAGLE MEDIUM “Nissui'8 (2)

[0090] FBS: Fetal Bovine Serum Certified, Origin: United States

[0091] PBS(−): Dulbecco's PBS(−) “Nissui”

[0092] The results are shown in Table 2. TABLE 2 Melanin ProductionSuppressing Activity of The Present Compound Melanin amount (% ofcontrol) Control 100.00 Kojic acid 0.1 mM 92.70 Kojic acid 0.3 mM 88.04Kojic acid 1 mM 68.80 Kojic acid 10 mM 18.73 Arbutin 0.1 mM 97.29Arbutin 0.3 mM 88.86 Arbutin 1 mM 83.02N-(6,8-dimercaptooctanoyl)aminoethanesulfonic acid Na—Zn 78.33 0.03 mMN-(6,8-dimercaptooctanoyl)aminoethanesulfonic acid Na—Zn 30.01  0.1 mMN-(6,8-dimercaptooctanoyl)aminoethanesulfonic acid Na—Zn 12.28  0.3 mMN-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid Na—Zn 64.43 0.03 mMN-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid Na—Zn 27.69  0.1 mMN-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid Na—Zn 9.85  0.3 mMN-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid Na—Zn 61.18 0.03 mMN-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid Na—Zn 29.20  0.1 mMN-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid Na—Zn 9.73  0.3 mMN-(6,8-dimercaptooctanoyl)methionine Zn 0.03 mM 63.05N-(6,8-dimercaptooctanoyl)methionine Zn 0.1 mM 50.18N-(6,8-dimercaptooctanoyl)methionine Zn 0.3 mM 16.41N-(6,8-dimercaptooctanoyl)phenylalanine Na—Zn 0.03 mM 71.43N-(6,8-dimercaptooctanoyl)phenylalanine Na—Zn 0.1 mM 37.78N-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn 0.03 mM 72.47N-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn 0.1 mM 47.64N-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn 0.3 mM 16.74

[0093] As evident from Table 2,N-(6,8-dimercaptooctanoyl)aminoethanesulfonic acid Na—Zn,N-(6,8-dimercaptooctanoyl)aminohexanoic acid Na—Zn,N-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid Na—Zn,N-(6,8-dimercaptooctanoyl)methionine Zn,N-(6,8-dimercaptooctanoyl)phenylalanine Na—Zn, andN-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn exhibited potentsuppressive action on melanin production, which was significantly morepotent than arbutin or kojic acid, compounds so far used as whiteningagent.

Test Example 3 Elastase Inhibiting Activity of The Present Compound TestMethod

[0094] 96-well plates were used. 1 mM of Suc(Ome)-Ala-Ala-Pro-Val-MCA,30 μl of a test sample, 210 μl of Tris-NaCl Buffer (25° C., pH 7.5) and30 μl of 1 unit/ml elastase were added and fluorescence was measuredevery minute (Ex. 360/40 nm, Em. 460/40 nm). Inhibition rates ofelastase activity were determined based on a calibration curve producedwith AMC (7-amino-4-methyl-coumarin) reference standard.

[0095] The results are shown in Table 3. TABLE 3 Esterase InhibitingActivity of The Present Compound Elastase inhibition rate (%) Elastinal0.03 mM 25.29 Elastinal 0.1 mM 56.87 Elastinal 0.3 mM 77.45 Elastinal 1mM 93.01 α-lipoic acid 0.03 mM −2.94 α-lipoic acid 0.1 mM 11.76 α-lipoicacid 0.3 mM 10.29 N-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid Na—Zn94.74 0.03 mM N-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid Na—Zn78.95 0.03 mM N-(6,8-dimercaptooctanoyl)methionine Zn 0.03 mM 7.41N-(6,8-dimercaptooctanoyl)methionine Zn 0.1 mM 68.52N-(6,8-dimercaptooctanoyl)methionine Zn 0.3 mM 88.89N-(6,8-dimercaptooctanoyl)phenylalanine Na—Zn 0.03 mM 83.33N-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn 0.03 mM 24.07N-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn 0.1 mM 48.15N-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn 0.3 mM 72.22

[0096] As evident from Table 3, elastase inhibiting activity comparableto Elastinal was noted with N-α-(6,8-dimercaptooctanoyl)-6-aminohexanoicacid Na—Zn, N-α-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid Na—Zn,N-α-(6,8-dimercaptooctanoyl)methionine Zn,N-α-(6,8-dimercaptooctanoyl)phenylalanine Na—Zn,N-α-(6,8-dimercaptooctanoyl)anthranilic acid Na—Zn. On the other hand,α-lipoic acid did not show any elastase inhibiting activity.

[0097] From the results, the present compound was found to be useful asan anti-wrinkle agent.

Preparation Example 1 Injection

[0098] The compound of Example 1  2.0 mg D-mannitol  5.0 g Distilledwater for injection Total amount  100 ml

[0099] Using the above ingredients, an injection is prepared by aconventional method.

Preparation Example 2 Eye Drops

[0100] The compound of Example 2  0.5 g Sodium chloride  0.5 g Boricacid  0.7 g Borax  0.3 g Methyl p-hydroxybenzoate 0.026 g Propylp-hydroxybenzoate 0.014 g Sterile purified water Total amount   100 ml

[0101] Using the above ingredients, eye drops are prepared by aconventional method.

Preparation Example 3 Tablets

[0102] The compound of Example 1 50 mg Lactose 80 mg Potato starch 17 mgPolyethylene glycol  3 mg

[0103] Using the above ingredients as the materials for one tablet,tables are prepared by a conventional method.

Preparation Example 4 Cosmetic Cream

[0104] The compound of Example 7, 8 or 9 0.5 g Stearic acid 2.0 gStearyl alcohol 7.0 g Squalane 5.0 g Octyldecanol 6.0 g Polyoxyethylenecetyl ether 3.0 g Glycerol monostearate 2.0 g Propylene glycol 5.0 gMethyl p-hydroxybenzoate 0.2 g Propyl p-hydroxybenzoate 0.1 g Sterilepurified water 73.7 g 

[0105] The above ingredients are mixed to form a cosmetic cream.

Industrial Applicability

[0106] The metal chelate compound of the present invention or apharmacologically acceptable salt thereof has tyrosinase inhibitingactivity, melanin production suppressing activity and elastaseinhibiting activity, and therefore is useful as an agent for prophylaxisand treatment of blotches and freckles and suntan of the skin, awhitening agent, a skin-beautifying agent and an anti-wrinkle agent.

[0107] Furthermore, the metal chelate compound of the present inventionis also useful for prophylaxis and treatment of a variety of disordersinduced by oxidative stress, such as ischemic cardiovascular disease,cerebral ischemia, arteriosclerosis, diabetes mellitus and cataract.

[0108] Some of the embodiments of the present invention are described indetail above. However, as various modifications and changes can be madeby those who skilled in the art to the specific embodiments withoutsubstantially departing from the novel disclosure and benefit of thepresent invention, all of such modifications and changes also fallwithin the spirit and scope of the present invention defined by thefollowing claims.

[0109] The present application is based on Japanese patent applicationNo. 2001-078571, filed in Japan, and all the contents thereof areincluded in the present application.

1. A N-(6,8-dimercaptooctanoyl)amino acid metal chelate compoundrepresented by the following formula (I),

wherein M denotes a metal, and A denotes an amino acid which is boundvia N, or a pharmacologically acceptable salt thereof.
 2. TheN-(6,8-dimercaptooctanoyl)amino acid metal chelate compound of claim 1or a pharmacologically acceptable salt thereof, wherein theN-(6,8-dimercaptooctanoyl)amino acid metal chelate compound is selectedfrom the group consisting of N-(6,8-dimercaptooctanoyl)-α-amino acidmetal chelates, N-(6,8-dimercaptooctanoyl)-β-amino acid metal chelates,N-(6,8-dimercaptooctanoyl)-γ-amino acid metal chelates,N-(6,8-dimercaptooctanoyl)-δ-amino acid metal chelates, andN-(6,8-dimercaptooctanoyl)-ε-amino acid metal chelates.
 3. TheN-(6,8-dimercaptooctanoyl)amino acid metal chelate compound of claim 1or a pharmacologically acceptable salt thereof, wherein theN-(6,8-dimercaptooctanoyl)amino acid metal chelate compound is selectedfrom the group consisting ofN-(6,8-dimercaptooctanoyl)aminoethanesulfonic acid metal chelates,N-(6,8-dimercaptooctanoyl)glycine metal chelates,N-(6,8-dimercaptooctanoyl)-aspartic acid metal chelates,N-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid metal chelates,N-(6,8-dimercaptooctanoyl)-γ-amino-n-butyric acid metal chelates,N-(6,8-dimercaptooctanoyl)phenylalanine metal chelates,N-(6,8-dimercaptooctanoyl)anthranilic acid metal chelates,N-(6,8-dimercaptooctanoyl)methionine metal chelates, andN-(6,8-dimercaptooctanoyl)cysteine metal chelates.
 4. TheN-(6,8-dimercaptooctanoyl)amino acid metal chelate compound of claim 1wherein the metal is zinc or a pharmacologically acceptable saltthereof.
 5. A medicament comprising the compound of one of claims 1 to 4or a pharmacologically acceptable salts thereof.
 6. A tyrosinaseinhibiting agent comprising the compound of one of claims 1 to 4 or apharmacologically acceptable salt thereof.
 7. A melanin productionsuppressing agent comprising the compound of one of claims 1 to 4 or apharmacologically acceptable salt thereof.
 8. A prophylactic ortherapeutic agent for blotches and freckles or a suntan of the skincomprising the compound of one of claims 1 to 4 or a pharmacologicallyacceptable salt thereof.
 9. A whitening agent comprising the compound ofone of claims 1 to 4 or a pharmacologically acceptable salt thereof. 10.A skin-beautifying agent comprising the compound of one of claims 1 to 4or a pharmacologically acceptable salt thereof.
 11. An elastaseinhibiting agent comprising the compound of one of claims 1 to 4 or apharmacologically acceptable salt thereof.
 12. An anti-wrinkle agentcomprising the compound of one of claims 1 to 4 or a pharmacologicallyacceptable salt thereof.
 13. A cosmetic preparation comprising thecompound of one of claims 1 to 4 or a pharmacologically acceptable saltthereof.
 14. A method for inhibition of tyrosinase comprisingadministering to a human an effective amount of the compound of one ofclaims 1 to 4 or a pharmacologically acceptable salt thereof.
 15. Amethod for suppression of melanin production comprising administering toa human an effective amount of the compound of one of claims 1 to 4 or apharmacologically acceptable salt thereof.
 16. A method for prophylaxisof therapeutic treatment of blotches and freckles or a suntan of theskin comprising administering to a human an effective amount of thecompound of one of claims 1 to 4 or a pharmacologically acceptable saltthereof.
 17. A method for whitening the skin comprising administering toa human an effective amount of the compound of one of claims 1 to 4 or apharmacologically acceptable salt thereof.
 18. A method for beautifyingthe skin comprising administering to a human an effective amount of thecompound of one of claims 1 to 4 or a pharmacologically acceptable saltthereof.
 19. A method for inhibiting elastase comprising administeringto a human an effective amount of the compound of one of claims 1 to 4or a pharmacologically acceptable salt thereof.
 20. A method forprevention or therapeutic treatment of wrinkles comprising administeringto a human an effective amount of the compound of one of claims 1 to 4or a pharmacologically acceptable salt thereof.
 21. Use of the compoundof one of claims 1 to 4 or a pharmacologically acceptable salt thereoffor the manufacture of a medicament.
 22. Use of the compound of one ofclaims 1 to 4 or a pharmacologically acceptable salt thereof for themanufacture of a tyrosinase inhibiting agent.
 23. Use of the compound ofone of claims 1 to 4 or a pharmacologically acceptable salt thereof forthe manufacture of a melanin production suppressing agent.
 24. Use ofthe compound of one of claims 1 to 4 or a pharmacologically acceptablesalt thereof for the manufacture of a prophylactic or therapeutic agentfor blotches and freckles or a suntan of the skin.
 25. Use of thecompound of one of claims 1 to 4 or a pharmacologically acceptable saltthereof for the manufacture of a whitening agent.
 26. Use of thecompound of one of claims 1 to 4 or a pharmacologically acceptable saltthereof for the manufacture of a skin-beautifying agent.
 27. Use of thecompound of one of claims 1 to 4 or a pharmacologically acceptable saltthereof for the manufacture of a elastase inhibiting agent.
 28. Use ofthe compound of one of claims 1 to 4 or a pharmacologically acceptablesalt thereof for the manufacture of an anti-wrinkle agent.
 29. Use ofthe compound of one of claims 1 to 4 or a pharmacologically acceptablesalt thereof for the manufacture of a cosmetic preparation.